Around 46% of the Worlds population lives in areas where mosquito-borne diseases including malaria, filariasis, viral encephalitides, dengue and yellow fever are endemic. It was recently established that the insect pathogenic fungus Metarhizium has the potential to control adult mosquitoes in an urban setting, but would work much better if its potency is increased. Using the resources of a two year R21 grant we compared mortality and malaria sporozoite prevalence in mosquitoes infected with transgenic Metarhizium strains expressing different combinations of insecticidal and anti-plasmodial proteins. Using Metarhizium to deliver arthropod toxins reduced the time it took to kill and produced a 150-fold reduction in effective spore doses. We hypothesize that under field conditions this will greatly increase the % of mosquitoes picking up a lethal infection and the effective persistence of the biopesticide. In this application, we propose a close collaboration with mosquito control expert Abdoulaye Diabates and his colleagues at Center Muraz that will enable us to evaluate the potential of transgenic Metarhizium in an area of Burkina Faso with very high densities of the malaria vector Anopheles gambiae (up to 200 bites/person/night) and Plasmodium falciparum sporozoite rates around 3%. Worldwide, GMO (genetically manipulated organisms) projects go through a review process that includes semi-field studies in a contained near-natural environment as a prerequisite for an open field release. A permit has been obtained from the Burkina Faso Ministry of Scientific Research and Innovation to build and use a malaria sphere that will consist of experimental huts (mimicking traditional housing), sugar sources (plants) for adults and created breeding sites (plastic containers), enclosed in a greenhouse frame with walls of mosquito netting to allow exposure to ambient climate conditions and simulate a natural mosquito habitat. To establish the potential value of modified fungi we will use the sphere to assess the efficacy, survivability, and environmental risk posed by pathogens engineered to express arthropod toxins in comparison with a strain with wild type virulence to insects but expressing ant-malarial proteins that reduce the parasite burden in mosquitoes by 98%. Our data will support predictive mathematical modeling of possible trials and hence provide a rational for designing future trials. These studies will 1) develop tools and test genetically engineered fungi that have the potential to greatly reduce malaria prevalence; 2) generate evidence to inform policy and attract donor funding for an open field release which will test epidemiological and clinical impact, and 3) enable scientists from a developing country to become directly involved in evaluating the potential use and application of transgenic microbes for future disease control.